Electrochemiluminescence of rare earth metal chelates

ABSTRACT

Luminescent chemical reagents that include complexes of rare earth metals with ligands such as aromatic heterocyclic nitrogen-containing compounds and semi-aromatic oxygen-containing compounds are used to detect small quantities of complex substances such as pharmaceuticals, metabolites, and microorganisms in complex sample mixtures.

FIELD OF THE INVENTION

[0001] The present invention relates to luminescent metal chelate labelsfor use in qualitative and quantitative chemical analysis. Morespecifically, the present invention relates to the use of chemicalreagents that include complexes of rare earth metals with ligands suchas aromatic heterocyclic nitrogen-containing compounds and semi-aromaticoxygen-containing compounds to detect small quantities of complexsubstances such as pharmaceuticals, metabolites, and microorganisms.

BACKGROUND OF THE INVENTION

[0002] There is a continuous and expanding need for rapid, highlyspecific methods of detecting and quantifying chemical, biochemical, andbiological substances. Of particular value are methods for measuringsmall quantities of pharmaceuticals, metabolites, microorganisms, andother materials of diagnostic value. Examples of such materials includenarcotics and poisons, drugs administered for therapeutic purposes,hormones, pathogenic microorganisms and viruses, antibodies,metabolites, enzymes and nucleic acids.

[0003] The presence of these materials can often be determined bybinding methods which exploit the high degree of specificity whichcharacterizes many biochemical and biological systems. Frequently usedmethods are based on, for example, antigen-antibody systems, nucleicacid hybridization techniques, and protein-ligand systems. In thesemethods, the existence of a complex of diagnostic value is typicallyindicated by the presence or absence of an observable “label” which hasbeen attached to one or more of the complexing materials.

[0004] The specific labeling method chosen often dictates the usefulnessand versatility of a particular system for detecting a material ofinterest. A preferred label should be inexpensive, safe, and capable ofbeing attached efficiently to a wide variety of chemical, biochemical,and biological materials without changing the important bindingcharacteristics of those materials. The label should give a highlycharacteristic signal, and should be rarely, and preferably never, foundin nature. The label should be stable and detectable in aqueous systemsover periods of time ranging from hours to months. Detection of thelabel should be rapid, sensitive, and reproducible without the need forexpensive, specialized facilities or personnel. Quantification of thelabel should be relatively independent of variables such as temperatureand the composition of the mixture to be assayed. Most advantageous arelabels which can be used in homogeneous systems, i.e., systems in whichseparation of the complexed and uncomplexed labeled material is notnecessary. This is possible if the detectability of the label ismodulated when the labeled material is incorporated into a specificcomplex.

[0005] A wide variety of labels have been developed, each withparticular advantages and disadvantages. For example, radioactive labelsare quite versatile, and can be detected at very low concentrations.However, they are expensive, hazardous, and their use requiressophisticated equipment and trained personnel. Furthermore, thesensitivity of radioactive labels is limited by the fact that thedetectable event can, in its essential nature, occur only once perradioactive atom in the labeled material. Moreover. radioactive labelscannot be used in homogeneous methods.

[0006] Thus, there is wide interest in non-radioactive labels. Theseinclude molecules observable by spectrophotometric, spin resonance, andluminescence techniques, as well as enzymes which produce suchmolecules. Among the useful non-radioactive labeling materials areorganometallic compounds. Because of the rarity of some metals inbiological systems, methods which specifically assay the metal componentof the organometallic compounds can be successfully exploited. Forexample, Cais, U.S. Pat. No. 4,205,952 (1980) discloses the use ofimmunochemically active materials labeled with certain organometalliccompounds for use in quantitating specific antigens. Any general methodof detecting the chosen metals can be used with these labels, includingemission, absorption and fluorescence spectroscopy, atomic absorption,and neutron activation. These methods often suffer from lack ofsensitivity, can seldom be adapted to a homogeneous system, and as withatomic absorption; sometimes entail destruction of the sample.

[0007] Of particular interest are labels which can be made to luminescethrough photochemical, chemical, and electrochemical means.“Photoluminescence” is the process whereby a material is induced toluminesce when it absorbs electromagnetic radiation. Fluorescence andphosphorescence are types of photoluminescence. “Chemiluminescent”processes entail the creation of the luminescent species by a chemicaltransfer of energy. “Electrochemiluminescence” entails the creation ofthe luminescent species electrochemically.

[0008] These luminescent items are of increasing importance. Forexample, Mandle, U.S. Pat. No. 4,372,745 (1983) discloses the use ofchemiluminescent labels in immunochemical applications. In the disclosedsystems, the labels are excited into a luminescent state by chemicalmeans such as by reaction of the label with H₂O₂ and an oxalate. Inthese systems, H₂O₂ oxidatively converts the oxalate into a high energyderivative, which then excites the label. This system will, inprinciple, work with any luminescent material that is stable in theoxidizing conditions of the assay and can be excited by the high energyoxalate derivative. Unfortunately, this very versatility is the sourceof a major limitation of the technique; typical biological fluidscontaining the analyte of interest also contain a large number ofpotentially luminescent substances that can cause high background levelsof luminescence.

[0009] Another example of the immunochemical use of chemiluminescencewhich suffers from the same disadvantages is Oberhardt et al., U.S. Pat.No. 4,290,815, (1981) who disclose the in situ electrochemicalgeneration of an oxidant (e.g., H₂O₂) in close proximity to animmunoreactant labeled with a chemiluminescent species. Theelectrogenerated oxidant diffuses to the chemiluminescent species andchemically oxidizes it, resulting in the net transfer of one or moreelectrons to the electrogenerated oxidant. Upon oxidation, thechemiluminescent species emits a photon. In contrast, the subjectinvention requires the direct transfer of electrons from a source ofelectrochemical energy to a chemiluminescent species which is capable ofrepeatedly emitting photons.

[0010] The present invention is concerned with electrochemiluminescentlabels. Suitable labels comprise electrochemiluminescent compounds,including organic compounds and organometallic compounds.Electrochemiluminescent methods of determining the presence of labeledmaterials are preferred over other methods for many reasons. They arehighly diagnostic of the presence of a particular label, sensitive,nonhazardous, inexpensive, and can be used in a wide variety ofapplications.

[0011] Organic compounds which are suitable electrochemical labelsinclude, for example, rubrene and 9,10-diphenyl anthracene. Manyorganometallic compounds are suitable electrochemical labels. Forinstance, Bard et al., in U.S. Pat. No. 5,310,687 (1994), discloses thata wide variety of analytes of interest and chemical moieties that bindto analytes of interest may be conveniently attached to ruthenium- orosmium-containing labels through amide or amine linkages. The labeledmaterials may then be determined by any of a wide variety of means,including photoluminescent, chemiluminescent, andelectrochemiluminescent means. It is also disclosed therein thatelectrochemiluminescent labels, including ruthenium- andosmium-containing labels and organic molecules such as rubrene and9,10-diphenyl anthracene, are particularly versatile and advantageous.

[0012] The series of elements having Atomic Numbers in the range 57-71are known as the lanthanides; they are: cerium, dysprosium, erbium,europium, gadolinium, holmium, lanthanum, lutetium, neodymium,praseodymium, promethium, terbium, thulium, and ytterbium. Thelanthanides are classically referred to as the ‘rare earths’. They arealso referred to as the inner-transition elements, because outerelectron structure is identical across the group; their electronstructures differ only at an inner level. Since the electronic diversitybetween the atoms is at some depth, the elements are very similarchemically. All of the lanthanides form trivalent ions and complexes.Their absorption bands are narrow compared with those of the normaltransition ions. Complexing agents, which alter the absorption spectraof normal transition ions by modifying their outer electron structures,have little effect on the lanthanide ions. In spite of a high charge,lanthanide ions are too large to cause significant polarization, socomplex formation is not facile.

[0013] With the transition metal chelates, the center metal atoms areunfulfilled d-orbitals. Under the ligand field, d-orbitals interact withthe ligand field leading to splitting the d-orbitals into two differentenergy levels, which correspond to the ground state and to an excitedstate. Unlike the case with transition metals, rare earth metals haveunfulfilled f-orbitals. The emission of rare earth metal chelates is dueto the intromolecular energy transfer from the ligand to the metal ion.Therefore, the emission is more characteristic of the metal than of theinteraction between the metal and its ligands.

[0014] The rare earth metal chelated usually have eight coordinationsites rather than six as for transition metal chelates. The eightcoordination sites are arranged as four in one plane and the other fourin another plane, with the metal located between the planes. However,the two planes twist 45° with respect to each other, so this type ofcompound is not of an octahedral configuration.

[0015] The Bard et al. chemical moieties as disclosed in U.S. Pat. No.5,310,687 do have one characteristic that constitutes a drawback incertain circumstances. The emission spectra of their chelates has a bandwidth on the order of 100 nm. This can make signal discriminationdifficult in multiple wavelength electrochemiluminescence measurements.Thus there remains a need for improvement in this area.

SUMMARY OF THE INVENTION

[0016] It has now been discovered that rare earth metal chelates may beprepared that have emission spectra band widths of less than 50 nm. Thegreat advantages of the use of the novel labeled materials based uponthe rare earth metal chelates, and of the methods of detecting them,follows.

[0017] According to the present invention there is provided a chemicalmoiety having the formula [MPL¹L²-(-link-)-]_(t)B wherein M is alanthanide element; P is a polydentate ligand of M; L¹ and L² areligands of M, each of which may be a substance covalently bound to oneor more of P, L¹, or L² through one or more covalent bond linkages suchas amide and amine linkages, said linkages designated as (-link-) andlinking B with at least one of P, L¹, or L²; t is an integer equal to orgreater than 1; and B is a substance of interest.

[0018] The present invention provides compounds particularly suitable asintermediates for attaching a luminescent lanthanide-containing label toamino groups of chemical, biochemical, and biological substances. Theseintermediates are thus particularly suitable for creating chemicalmoieties according to the present invention. The intermediates are themono- and di-N-hydroxysuccinimide esters of lanthanidebis(2,2′-bipyridine)(2,2′-bipyridine-4,4′-dicarboxylic acid) and theirsalts and ruthenium or osmiumbis(2,2′-bipyridine)(4,4′-di(chloromethyl)-2,2′-bipyridine). Thesecompounds are generally synthesized by procedures which are known in theart.

[0019] The present invention provides methods for determining thepresence of the novel chemical moieties. The present invention alsoprovides methods of determining the presence of a chemical moiety asdescribed above. The methods comprise:

[0020] a) forming a reagent mixture under suitable conditions containingthe chemical moiety;

[0021] b) inducing the moiety to emit electromagnetic radiation byexposing the reagent mixture to chemical energy or electrochemicalenergy; and

[0022] c) detecting the emitted electromagnetic radiation and therebydetermining the presence of the chemical moiety.

[0023] This invention further provides for the use oflanthanide-containing labels in binding methods for determining thepresence of substances of interest. These methods may be used todetermine labeled moieties of interest, to employ labeled moieties todetermine analytes of interest, or to use labelled analogues of analytesof interest to determine analytes of interest in competitive bindingassays. These binding methods may be homogeneous or heterogeneousbinding methods.

[0024] Still further, the present invention provides systems fordetermining the presence of the lanthanide-containing chemical moietiesof this invention. These systems comprise a means for inducing thechemical moiety to emit electromagnetic radiation.

[0025] The present invention also provides for use ofelectrochemiluminescent labels in binding methods for determining thepresence of substances of interest. These methods can be used todetermine labeled moieties of interest, to employ labeled moieties todetermine analytes of interest, or to use labeled analogues of analytesof interest to determine analytes of interest in competitive bindingassays. These binding methods can be homogeneous or heterogeneousbinding methods.

[0026] A specific embodiment of the invention provides for compositionswhich contain two or more different chemical moieties. Each of themoieties may be chemical species which can be induced to emitelectromagnetic radiation of different wavelength. In another embodimentof the invention the chemical moieties may be chemical species each ofwhich is induced to emit electromagnetic radiation by exposure to energyof a different value or from a different source. A different substanceor analyte of interest may then be specifically attached to each of thedifferent chemical moieties. By using these compositions and methods itis possible to determine two or more different substances or analytes ofinterest that may be present in the sample under examination.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1(a) shows a cyclic voltammogram for 2 mM europium chelate in0.1 M tetrabutylammonium hexafluorophosphate (hereinafter “TBAPF₆”)acetonitrile solution.

[0028]FIG. 1(b) shows a cyclic voltammogram for 2 mM europium chelatewith 10 mM tripropylamine (hereinafter “TPA”) added in 0.1 M TBAPF₆acetonitrile solution.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The Chemical Moiety: M and Its Ligands

[0030] According to the present invention, there is provided a chemicalmoiety having the formula

[MPL¹L²-(-link-)-]_(t)B

[0031] wherein M is a lanthanide; P is a polydentate ligand of M; L¹ andL² are ligands of M, each of which may be a substance covalently boundto one or more of P, L¹, or L² through one or more amide or aminelinkages; t is an integer equal to or greater than 1; P, L¹, L², and Bare of such number that the total number of bonds to M provided by theligands of M equals the coordination number of M; (-link-) designatesthe bond or bonds that connect B to P, L¹, and L²; and P, L¹, L², and Bare of such composition that the chemical moiety can be induced to emitelectromagnetic radiation.

[0032] In accordance with the present invention, M may be cerium,dysprosium, erbium, europium, gadolinium, holmium, lanthanum, lutetium,neodymium, praseodymium, promethium, terbium, thulium, or ytterbium. Inthe currently preferred embodiments of the present invention, M iscerium, europium, terbium, or ytterbium, with europium being mostpreferred.

[0033] The chemical moiety in accordance with the present inventionshould have at least one polydentate ligand of M. When the moiety hasmore than one polydentate ligand, the polydentate ligands may be thesame or different. Polydentate ligands include aromatic and aliphaticligands. Suitable aromatic polydentate ligands include aromaticheterocyclic ligands such as, for example, bipyridyl, bipyrazyl,terpyridyl, phenanthrolyl, and porphyrins, as well as semi-aromaticoxygen-containing compounds such as, for example, diketone-typecompounds. The semi-aromatic oxygen-containing compounds are currentlypreferred.

[0034] Suitable polydentate ligands may be unsubstituted, or substitutedby any of a large number of substituents known to the art. Suitablesubstituents include for example, alkyl, substituted alkyl, aryl,substituted aryl, aralkyl, substituted aralkyl, carboxylate,carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureido, maleimide,sulfur-containing groups, phosphorus containing groups, and thecarboxylate ester of N-hydroxysuccinimide.

[0035] Additionally, at least one of L¹ and L² may be a polydentatearomatic heterocyclic ligand that contains nitrogen as well assemi-aromatic oxygen-containing compounds such as, for example,diketone-type compounds. The semi-aromatic oxygen-containing compoundsare currently preferred. Suitable polydentate ligands include, but arenot limited to, bipyridyl, bipyrazyl, terpyridyl, phenanthroyl, aporphyrin, substituted bipyridyl, substituted bipyrazyl, substitutedterpyridyl, substituted phenanthroyl, or a substituted porphyrin. Thesenitrogen-containing aromatic ligands as well the semi-aromaticoxygen-containg ligands may be substituted with an alkyl, substitutedalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureido, maleimide,a sulfur-containing group, a phosphorus-containing group, or thecarboxylate ester of N-hydroxysuccinimide.

[0036] In one embodiment of the invention the chemical moiety containstwo bidentate ligands, each of which is bipyridyl, bipyrazyl,terpyridyl, phenanthrolyl, substituted bipyridyl, substituted bipyrazyl,substituted terpyridyl, or substituted phenanthrolyl. In anotherembodiment of the invention the chemical moiety contains three bidentateligands, each of which is bipyridyl, bipyrazyl, terpyridyl,phenanthrolyl, substituted bipyridyl, or substituted phenanthrolyl, forinstance

[0037] This chemical moiety may have one or more monodentate ligands, awide variety of which are known to the art. Suitable monodentate ligandsinclude, for example, carbon monoxide, cyanides, isocyanides, halides,and aliphatic, aromatic and heterocyclic phosphines, amines, stibines,and arsines.

[0038] In still another embodiment of the invention the chemical moietycontains a tetradentate ligand such as a porphyrin or substitutedporphyrin, or mixed nitrogen-containing and oxygen-containing ligands,for instance

[0039] In a preferred embodiment of the invention, the chemical moietycontains three semi-aromatic oxygen-containing diketone-type ligands.

[0040] Preferred embodiments of this chemical moiety comprisebis(2,2′-bipyridyl)europium(II), tris(2,2′-bipyridyl)europium(II),europium tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate),which has the structure

[0041] and terbium tris(2,2,6,6-tetramethyl-3,5-heptanedionate), whichhas the structure

[0042] Some of these chelates are known. The others can be prepared byprocedures analogous to those used for preparing the known species. U.S.Pat. No. 3,700,410 discloses the preparation of certain preferredchelates that may be used in accordance with the present invention. Theentire disclosure of that patent is expressly incorporated herein byreference.

[0043] It is within the scope of the invention for one or more of theligands of M to be attached to additional chemical labels, such as, forexample, radioactive isotopes, fluorescent components, or additionalluminescent europium-containing centers.

[0044] The Chemical Moiety: Biological and Nonbiological Substances B

[0045] Suitable substances (B) include many biological substances, forexample, whole cells, viruses, subcellular particles, proteins,lipoproteins, glycoproteins, peptides, nucleic acids polysaccharides,lipopolysaccharides, lipids, fatty acids, cellular metabolites,hormones, pharmacological agents, tranquilizers, barbiturates,alkaloids, steroids, vitamins, amino acids and sugars. Whole cell may beanimal, plant, or bacterial, and may be viable or dead. Examples includeplant pathogens such as fungi and nematodes. Within this application theterm “subcellular particles” means subcellular organelles, membraneparticles as from disrupted cells, fragments of cell walls, ribosomes,multienzyme complexes, and other particles which can be derived fromliving organisms. Also, within this application, nucleic acids meanschromosomal rNA, plasmid rNA, viral rNA, and recombinant INA derivedfrom multiple sources. Nucleic acids also include RiAs, for examplemessenger RiAs, ribosomal IRNAs and transfer RNAS. Polypeptides include,for example, enzymes, transport proteins, receptor proteins andstructural proteins such as viral coat proteins. Preferred polypeptidesare enzymes and serum-derived antibodies. Particularly preferredpolypeptides are monoclonal antibodies. Thus, in one embodiment of theinvention, B is a nucleotide or a polynucleotide. In another embodiment,B is a serum-derived antibody or a monoclonal antibody. Hormonesinclude, for example, insulin and T4 thyroid hormone. Pharmacologicalagents include, for example, cardiac glycosides. It is also within thescope of this invention of include synthetic nucleic acids, andsynthetic membranes, vesicles and liposomes. The foregoing is notintended to be a comprehensive list of the biological substancessuitable for use in this invention, but is meant only to illustrate thewide scope of the invention.

[0046] It is within the scope of this invention to include labelednonbiological substances, including polymeric materials. Thesesubstances may be in the form of soluble polymeric molecules, or any ofthe large variety of known macroscopic forms such as, for example,beads, or containers such as test tubes, bottles, assay wells or thelike.

[0047] The Chemical Moiety: Linkages

[0048] Biological and nonbiological substances (B) are covalently boundto a ligand of M through one or more covalent linkages. These aredesignated as (-link-) in the formula set forth above and in the claims.Any type of covalent attachment is intended to be covered by the generalterm (-link-). Amide and amine linkages are preferred. In the case ofamide linkages, the linkages may be oriented so that material (B) isbonded directly either to the carbonyl or to the nitrogen of the amidelinkage. These chemical moieties may be ionized. If so, it is understoodin the art that many different counterions will serve to neutralize thecharge of preparations of the chemical moiety. Suitable cations include,for example, H⁺, NH₄ ⁺ guanidinium, Ag⁺, Li⁺, N⁺, K⁺, Mg²⁺, and Mn²⁺.Suitable anions include, for example, halides, OH⁻, carbonate, SO₄ ²⁻,hexafluorophosphate and tetrafluoroborate.

[0049] It is also within the scope of this invention for the labeledsubstance (B) to be labeled by greater than one, e.g., two, three, fouror more electrochemiluminescent centers.

[0050] Intermediates

[0051] The present invention also provides compounds that areparticularly suitable as intermediates for attaching a luminescenteuropium-containing label to amino groups of chemical, biochemical andbiological substances. These intermediates are thus particularlysuitable for synthesizing chemical moieties according to the presentinvention. They include compounds having the formula

MPL¹L²-(-link-)

[0052] wherein M is a lanthanide element; P is a polydentate ligand ofM; L¹ and L² are ligands of M, each of which may be a substancecovalently bound to one or more of P, L¹, or L² through one or morecovalent bond linkages, said linkages designated as (-link-) and beingcovalent bonds; P, L¹, and L² are of such number that the total numberof bonds to M provided by the ligands of M equals the coordinationnumber of M; and P, L¹, and L² are of such composition that the chemicalmoiety can be induced to emit electromagnetic radiation. In thesecompounds, (-link-) may contain activators such as N-hydroxysuccinimidylmoieties, amino moieties, or thiol moieties. Among the intermediates ofthe invention are the mono- and di-N-hydroxysuccinimide esters ofeuropium 4,4′(dicarboxy)-2,2′-bipyridyl, bis(2,2′-bipyridyl) and theirsalts; and europium 4,4′(dichloromethyl)-2,2′-bipyridyl,bis(2,2′bipyridyl) and their salts.

[0053] A preferred method of synthesizing the europium-containingN-hydroxysuccinimide esters is to first react europiumdichlorobis(2,2′-bipyridine) with 2,2′-bipyridine-4,4′-dicarboxylic acidin a hot aqueous methanol solution of sodium bicarbonate. Afteracidification, an aqueous solution of NAPF₆ is added to the solution ofcarboxylated europium compound. The isolated hexafluorophosphate salt ofthe europium complex is then esterified by reaction withN-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide indimethylformamide. Of course, many variations on the structure of theN-hydroxysuccinimide component are possible without substantiallyaltering the usefulness of the inventive intermediates.

[0054] These intermediates may be ionized. If so, it is understood inthe art that many different counterions will serve to neutralize thecharge of preparations of the intermediate and form a salt. Suitablecations for forming these salts include for example NH₄ ⁺, guandinium,Ag⁺, Li⁺, Na⁺, K⁻, Ca²⁺, Mg²⁺, Mn²⁺, and Cd²⁺. Suitable anions forforming these salts include, for example, halides, carbonate, SO₄ ²⁻hexafluorophosphate, and tetrafluoroborate.

[0055] These intermediates are useful for labeling substances containinga free amino group capable of attacking the carboxylate ester, andthereby displacing N-hydroxysuccinimide, or of attacking thechloromethyl group, and thereby displacing chloride. Use of theseintermediates to label analytes of interest is preferred overisothiocyanates of the prior art (e.g. Weber, U.S. Pat. No. 4,293,310).Isothiocyanates are generally prepared by reaction of a primary aminewith carbon disulfide or thiophosgene, each of which is volatile andhighly toxic. Carbon disulfide is also an acute fire and explosionhazard. The required precursor primary aromatic amines are moredifficult to obtain than the precursor aromatic carboxylic acids used inthe present invention. Also, the intermediates of the present inventionare less reactive and more easily stored and handled than theisothiocyanate derivatives.

[0056] Determining the Presence of Chemical Moieties: In General

[0057] The present invention provides methods for determining thepresence of the chemical moieties of the invention by forming a reagentmixture which comprises the chemical moiety and detecting the presenceof the moiety in such reagent mixture. As such throughout thisapplication, and as will be readily appreciated by those skilled in theart to which this invention pertains, “reagent mixture” includes any andall combinations of the chemical moieties with other substances orreagents which may be employed in the practice of this invention. Thespecific combination may be in the form of an aqueous or nonaqueoussolution, a suspension or emulsion, a solid or semisolid, or a gas,limited only by such limitations as may be imposed by the detectionmethod used to detect the presence of the chemical moiety. For instance,electrochemiluminescence identifications may be performed with europiumtris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate) inaqueous solution and with terbiumtris(2,2,6,6-tetramethyl-3,5-heptanedionate) in methyl chloride.

[0058] The chemical moieties may be detected by methods well known inthe art including, for example, emission and absorption spectroscopy,e.g. ultraviolet absorption, infrared absorption, and fluorescenceemissions; atomic absorption, electrochemical, e.g. anodic strippingvoltametry; neutron activation and chemical methods. Of particularinterest are photoluminescence, chemiluminescence andelectrochemiluminescence methods. In one embodiment of the invention,the presence of the chemical moiety may be determined by inducing thechemical moiety to emit electromagnetic radiation and detecting theemitted radiation. In another embodiment of the invention, the chemicalmoiety may be induced to emit electromagnetic radiation by exposing thereagent mixture to electromagnetic, chemical or electrochemical energy.In yet another embodiment of the invention, the chemical moiety may beinduced to emit electromagnetic radiation by exposing the reagentmixture to chemical or electrochemical energy.

[0059] Eu chelate may be determined at very low concentrations usingluminescence techniques. Applicants' experience with Eu chelate labeledsubstances indicates the advantages of using europium-containingcompounds as chemical labels. They are stable for long periods and maybe attached efficiently to a wide variety of chemical, biochemical andbiological materials. The labels are safe and relatively inexpensive.They give a highly characteristic signal and do not occur in nature.Measurements based on luminescence of the labels are sensitive, fast,reproducible and utilize simple instrumentation. There is very littleinterference with detection based on luminescence of these labels bysuch components as phosphate buffered saline, Tween (a surfactant),liver tissue extract or serum. Luminescence-based measurement of theselabels does not destroy the sample or labeled materials and may beperformed repetitively. The signal is generated repeatedly by eachmolecule of label, thereby enhancing the sensitivity with which theselabel may be detected. The presence of labeled materials may bedetermined qualitatively or quantitatively depending on the needs of theparticular application. The word “determined”, as used in this patentapplication, refers to either qualitative or quantitative determinationsof the labeled material.

[0060] Accordingly, this invention provides a method of determining thepresence of a chemical moiety having the formula:

[MPL¹L²-(-link-)-]_(t)B

[0061] wherein M is europium; P is a polydentate ligand of M; L¹ and L²are ligands of M, each of which may be the same as, or different fromeach other ligand; B is a substance which is a ligand of M or isattached to one or more of P, L¹, or L²; t is an integer equal to orgreater than 1; (-link-) designates a covalent bond or bonds whichconnect B to P, L¹, or L²; and P, L¹, L², L³, L⁴, L⁵ and L⁶ and B are ofsuch composition and number that the chemical moiety can be induced toemit electromagnetic radiation and the total number of bonds to Mprovided by the ligands of M equals the coordination number of M.

[0062] The method comprises:

[0063] a) forming a reagent mixture under suitable conditions containingthe chemical moiety;

[0064] b) inducing the moiety to emit electromagnetic radiation byexposing the reagent mixture to chemical energy or electrochemicalenergy; and

[0065] c) detecting the emitted electromagnetic radiation and therebydetermining the presence of the chemical moiety.

[0066] Suitable conditions for forming the reagent mixture will be knownto those skilled in the art and will depend on the type of reagentmixture involved. For example, suitable conditions for an aqueousreagent mixture may include appropriate concentrations of chemicalmoiety and other reagents such as oxidants, pH, salt concentration andthe like. For a solid sample, suitable conditions for forming a reagentmixture may include addition of a conducting liquid.

[0067] Determining the Presence of Bound Chemical Moieties

[0068] The methods of this invention include a method of determining thechemical moiety wherein the moiety is capable of binding to a chemicalagent, i.e. forming a specific complex with the chemical agent.

[0069] Suitable chemical agents include, but are not limited to, wholecells, viruses, subcellular particles, nucleic acids polysaccharides,proteins, glycoproteins, lipoproteins, lipopolysaccharides, lipids,fatty acids, peptides cellular metabolites, hormones, pharmacologicalagents, tranquilizers, barbiturates, alkaloids, steroids, vitamins,amino acids, sugars or non-biological polymers.

[0070] In one embodiment of the invention, the chemical agent may beimmobilized on the surface of an assay vessel. In another embodiment,the chemical agent may be a serum-derived antibody or a monoclonalantibody.

[0071] Of particular interest are antibody-antigen pairs of materials.This binding method may be used to determine the presence of labeledantigens, such as, for example, digoxin or digitoxin in complex mixturesby first exposing the mixture to immobilized antibodies specific for theantigen of interest, and then measuring the amount of labeled materialbound to the immobilized antibodies.

[0072] The invention further provides methods for determining thepresence of analytes of interest which bind to a chemical moiety, saidmoiety having the formula:

[MPL¹L²-(-link-)-]_(t)B

[0073] wherein M is europium; P is a polydentate ligand of M; L¹ and L²are ligands of M, each of which may be the same as, or different fromeach other ligand; B is a substance which is a ligand of M or isattached to one or more of P, L¹, or L²; t is an integer equal to orgreater than 1; (-link-) designates a covalent bond or bonds whichconnect B to P, L¹, or L²; and P, L¹, L², L³, L⁴, L⁵ and L⁶ and B are ofsuch composition and number that the chemical moiety can be induced toemit electromagnetic radiation and the total number of bonds to Mprovided by the ligands of M equals the coordination number of M.

[0074] The method comprises:

[0075] a) forming a reagent mixture under suitable conditions containingthe chemical moiety;

[0076] b) inducing the moiety to emit electromagnetic radiation byexposing the reagent mixture to chemical energy or electrochemicalenergy; and

[0077] c) detecting the emitted electromagnetic radiation and therebydetermining the analyte of interest.

[0078] Within this application “complementary material” means anysubstance capable of forming complexes with both an analyte of interestand a labeled analyte of interest or a labeled analogue of an analyte ofinterest.

[0079] Electromagnetic Radiation

[0080] The phrase “inducing to emit electromagnetic radiation” refers tocreating an excited states of said moiety which luminesces atwavelengths between 200 nanometers and 900 nanometers at ambienttemperatures. The present invention envisions lanthanide-containingmoieties such as europium-containing moieties and encompasses the widevariety of luminescent moieties which can be made by varying thechemical structure of the ligands. Each of these variations in the metaland the ligands can change the precise value of the energy inputrequired to create the luminescent excited state. Similarly, thewavelength of the emitted electromagnetic radiation will be dependentupon the nature and environment of the europium-containing material.Generally, photoluminescence excitation and emission will occur withelectromagnetic radiation of between about 200 nanometers and about 900nanometers in wavelength. Chemiluminescent and electrochemiluminescentemission will generally occur with the emitted electromagnetic radiationbeing between about 100 nanometers and about 1500 nanometers inwavelength, and preferably of wavelengths between 200 and 900nanometers. The potential at which the reduction or oxidation of thechemical moiety will occur depends upon its exact chemical structure aswell as factors such as the pH of the solution and the nature of theelectrode used. Generally, it is well known in the art how to determinethe optimal emission and excitation wavelengths in a photoluminescentsystem, and the optimal potential and emission wavelength of anelectrochemiluminescent and chemiluminescent system.

[0081] It should be clear that there are many methods for quantifyingthe amount of luminescent species present. The rate of energy input intothe system can provide a measure of the luminescent species. Suitablemeasurements include, for example, measurements of electric current whenthe luminescent species is generated electrochemically, the rate ofreductant or oxidant utilization when the luminescent species isgenerated chemically or the absorption of electromagnetic energy inphotoluminescent techniques. In addition, of course, the luminescentspecies can be detected by measuring the emitted electromagneticradiation. All of these measurements can be made either as continuous,rate-based measurements, or as cumulative methods which accumulate thesignal over a long period of time. An example of rate-based measurementsis the use of photomultiplier tubes, photodiodes or phototransistors toproduce electric currents proportional in magnitude to the incidentlight intensity. Examples of cumulative methods are the integration ofrate-based data, and the use of photographic film to provide cumulativedata directly.

[0082] All of these luminescence-based methods entail repeatedluminescence by the europium-containing compound. The repetitive natureof the detectable event distinguishes these labels from radioactiveisotopes or bound chemiluminescent molecules such a luminal. The latterlabels produce a detectable event only once per molecular (or atom) oflabel, thereby limiting their detectability.

[0083] Binding Methods

[0084] In the chemical moieties useful in these methods, biological andnonbiological substance (B) may be incorporated into the moieties bycoordination directly to M or by attachment to a ligand of M. Attachmentmay be through covalent bonding, or by electrostatic or hydrogenbonding. Many diverse means of effecting covalent bonding of substances(B) to ligands of M are available. The attaching linkage may be, forexample, an amide or amine bond, an ester or thioester, an ether orthioester, or any of many other means known to the art. The type oflinkage will be determined by the substituents of the ligand and thesuitable chemical groups available for binding with the ligand on thesubstance that is to be labeled.

[0085] The analyte of interest and the chemical moiety may be any pairof substances which are capable of binding together in a specificmanner. Such substances include for example, whole cells, viruses,subcellular particles, nucleic acids, polysaccharides, proteins,glycoproteins, lipoproteins, lipopolysaccharides, lipids, fatty acids,peptides, cellular metabolites, hormones, pharmacological agents,tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino acids,sugars and non-biological polymers. Of particular interest areantibody-antigen pairs. One embodiment of the invention provides the useof labeled antibodies to determine the presence of cell surfaceantigens, or to label particular cells for detection by cell sortingmethods. Antigens immobilized by, for example, attachment toimmobilized, unlabeled antibodies can be detected by labeled antibodiesin a method commonly known as a “sandwich” method.

[0086] In competitive binding assays, B may be the same substance as theanalyte of interest or an analogue of the analyte, and capable ofparticipating in the formation of a specific complex with acomplementary material. Such analytes and complementary materialsinclude, whole cells, viruses, subcellular particles, nucleic acids,polysaccharides, proteins, glycoproteins, lipoproteins,lipopolysaccharides, lipids, fatty acids, peptides, cellularmetabolites, hormones, pharmacological agents, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars andnon-biological polymers. Examples of such analytes and complementarymaterials include insulin, digoxin, digitoxin, T4 thyroid hormone, afungus or nematode, a serum-derived antibody or a monoclonal antibody, aDNA fragment or an RNA fragment of particular interest areantibody-antigen based methods. These methods “are analogous to the wellknown radioimmunoassay, wherein an analyte of interest is detected whenit displaces a radioactive analogue of the analyte from an antibody. Themarry variations on radioimmunoassay known to the art can, in principle,be used to advantage by employing moieties labeled according to thepresent invention in place of radioactively labeled compounds.

[0087] The present invention further provides heterogeneous andhomogeneous binding methods which utilize the chemical moieties providedherein. In heterogeneous binding methods, the bound labeled substancemust be physically separated from the unbound labeled substance beforemeasurement of the presence of label. This is frequently accomplished inantibody-antigen systems by immobilizing one component, the antibody forexample, by attachment to an insoluble matrix such as a filter or to thesurface of beads or reaction vessels such as test tubes. Theantigen-containing solution is poured through the filter or into thereaction vessel, and then washed away from the filter or sides of thereaction vessel. Only antigen specifically bound to antibody will remainto be determined.

[0088] In homogeneous methods, by contract, the bound and unboundlabeled material are present in the same reaction mixture when thepresence of label is measured. This is possible when binding modifiesthe properties of the signal detectable from the label. There are manyways that luminescent labels can be used in homogeneous systems. Forexample, binding of the analyte to the chemical moiety can directlyinfluence the signal detectable from the label. Additionally, aluminescence quencher may be positioned on an antibody so that bindingof a labeled antigen to the antibody could result in suppression of theluminescence of the label by the luminescence quencher on the antibody.Many homogeneous methods for luminescent labels are known to the art;and some of them are reviewed in Boguslaski and Li (1982), “HomogeneousImmunoassays,” Applied Biochemistry and Biotechnology, 7, pp. 401-414.

[0089] In one embodiment of the invention, the analyte is fixed to aninsoluble matrix. Such a method may be performed as a sandwich assayi.e. the chemical moiety becomes bound to the immobilized analyte andunbound moiety is washed away from the matrix.

[0090] Another embodiment comprises a chemical agent to which the moietyis capable of binding being fixed to an insoluble matrix and thechemical moiety being a component of a biological fluid or syntheticreaction.

[0091] Additionally, the competitive binding methods of the presentinvention may comprise the complementary material being fixed to aninsoluble matrix.

[0092] Both the heterogeneous and homogeneous competitive methods of thepresent invention comprise the complementary material being monoclonalantibody and the insoluble matrix being the surface of an assay vessel.

[0093] Inducing the Emission of Electromagnetic Radiation

[0094] The methods of the present invention may be performed by exposingthe reagent mixture to electrochemical energy or to chemical energy.Additionally, the reagent mixture may be exposed to a combination ofelectromagnetic radiation, chemical energy, and electrochemical energy.

[0095] The chemical moiety may be oxidized by exposure to an energysource. Such an energy source amy be a chemical oxidizing agent.Examples of such oxidizing agents include CE(IV) salts or PbO₂.Furthermore, the chemical moiety may be reduced by exposure to an energysource. Such an energy source may be a chemical reducing agent. Anexample of a suitable reducing agent is magnesium.

[0096] The methods of the present invention may comprise inducing thechemical moieties to emit electromagnetic radiation more than once.

[0097] The reagent mixture may comprise oxalate, pyruvate, lactate,malonate, citrate, tartrate or peroxydisulfate. Furthermore, thechemical moiety may be reduced by exposure to an energy source and thereagent mixture may comprise peroxydisulfate. Moreover, the chemicalmoiety may be oxidized by exposure to an energy source and the reagentmixture may comprise oxalate, pyruvate, lactate, malonate, citrate ortartrate.

[0098] Methods of detecting the chemical moiety are provided wherein thereagent mixture is continuously exposed to electrode whose potentialoscillates between a potential sufficient to effect the reduction ofsaid chemical moiety and a potential sufficient to effect the oxidationof the chemical moiety.

[0099] The chemical moiety may be oxidized by exposure to an electrodewhose potential oscillates above and below a potential sufficient tooxidize the chemical moiety, the reagent mixture comprising oxalate,pyruvate, lactate, malonate, tartrate or citrate. Moreover, the chemicalmoiety may be oxidized by exposure to an electrode whose potential isconstant and sufficient to oxidize it, the reagent mixture comprisingoxalate, pyruvate, lactate, malonate, tartrate or citrate.

[0100] The chemical moiety may also be reduced by exposure to anelectrode whose potential oscillates above and below a potentialsufficient to reduce it, the reagent mixture comprising peroxydisulfate.Such reagent mixture may additionally comprise acetonitrile.Furthermore, the chemical moiety may be reduced by exposure to anelectrode whose potential is constant and sufficient to reduce it, thereagent mixture comprising peroxydisulfate. Such reagent mixture mayalso comprise acetonitrile.

[0101] When the chemical moiety is exposed to electrochemical orchemical energy, the emitted electromagnetic radiation may becontinuously detected. Such electromagnetic radiation may be detectedvisually or with a photodiode. Furthermore, when the chemical moiety isexposed to electrochemical or chemical energy, the emitted radiation maybe detected cumulatively, e.g. with a photographic film.

[0102] U.S. Pat. No. 5,147,806 (Kamin et al.) discloses a method andapparatus for conducting electrochemiluminescence measurements. Themethod and apparatus control the initial conditions relating to thesurface state of a triggering working electrode by reproducibly creatingand maintaining a favorable surface condition. This enhances theprecision and detection limit of the measurements. Assays are performedwith electrochemiluminescence detection in a flow-through cellenvironment. The precision and detection limit are enhanced byalternating initialization and measurement steps. The entire disclosureof the Kamin et al. patent is expressly incorporated herein byreference.

[0103] Means and Systems

[0104] The present invention also provides a system for determining thepresence of a chemical moiety having the formula:

[MPL¹L²-(-link-)-]_(t)B

[0105] wherein M is europium; P is a polydentate ligand of M; L¹ and L²are ligands of M, each of which may be the same as, or different fromeach other ligand; B is a substance which is a ligand of M or isattached to one or more of P, L¹, or L²; t is an integer equal to orgreater than 1; and P, L¹, L², L³, L⁴, L⁵ and L⁶ and B are of suchcomposition and number that the chemical moiety can be induced to emitelectromagnetic radiation and the total number of bonds to M provided bythe ligands of M equals the coordination number of M.

[0106] The system comprises:

[0107] a) a reagent mixture comprising the chemical moiety;

[0108] b) means for inducing the chemical moiety to emit electromagneticradiation; and

[0109] c) means for detecting the emitted electromagnetic radiation.

[0110] A system for determining the presence of an analyte of interestwhich binds to a chemical moiety is also provided, wherein the moietyhaving the structural formula:

[MPL¹L²-(-link-)-]_(t)B

[0111] is used, with the components of the structure being the same asthose described above. This system comprises:

[0112] a) the chemical moiety;

[0113] b) a means for contact the chemical moiety with the analyte ofinterest to form a reagent mixture;

[0114] c) a means for inducing the chemical moiety to emitelectromagnetic radiation; and

[0115] d) a means for detecting the emitted electromagnetic radiation.

[0116] Electromagnetic Radiation of Differing Wavelengths

[0117] This invention also concerns compositions which comprise theeuropium-containing chemical moieties of this invention and one or moredifferent chemical moieties each of which can be induced to emitelectromagnetic radiation of a different distinct wavelength. Thesecompositions are useful in methods and systems of detecting two or moredifferent substances or analytes of interest contained in a mixture ofthe same and other substances.

[0118] In one embodiment of the inventions the chemical moieties areeach attached to different analytes of interest.

[0119] The different chemical moiety or moieties may be any suitablechemical moiety such as inorganic, organic or organometallic compoundswhich can be induced to emit electromagnetic radiation, e.g. rubrene or9,10-diphenylanthracene. These moieties may be such moieties that areinduced to emit electromagnetic radiation when exposed to energy ofdifferent values or sources than the energy used to induceelectromagnetic radiation from the europium-containing chemicalmoieties. In a specific embodiment of the invention, each other chemicalmoiety emits electromagnetic radiation of a different distinctwavelength when induced to emit electromagnetic radiation by energy ofthe same source and value that induces the europium-containing chemicalmoiety to emit electromagnetic radiation.

[0120] Methods for determining these chemical moieties comprise forminga reagent mixture under suitable conditions containing the chemicalmoieties and then inducing the chemical moieties to emit electromagneticradiation by exposing the reagent mixture to chemical energy orelectrochemical energy. The presence of each of the moieties isdetermined by detecting the electromagnetic radiation of differentwavelengths emitted by each of the moieties.

[0121] The invention also concerns a method of determining the presenceof one or more analytes of interest which bind selectively to differentchemical moieties present in the same mixture. The method comprisescontacting the analytes with the chemical moieties under suitableconditions so as to form a reagent mixture. The moieties are induced toemit electromagnetic radiation by exposing the reagent mixture tochemical energy or electrochemical energy and the emittedelectromagnetic radiation of different wavelengths is detected todetermine the presence of each of the analytes of interest.

[0122] These methods in which the presence of two or more chemicalmoieties is determined in a mixture are applicable to all instancesdescribed previously for determining the europium-containing luminescentlabels. This embodiment, however, allows for the determination of two ormore different substances present in the same sample simultaneously.

[0123] Also provided are systems for determining the presence of one ormore different chemical moieties or analytes of interest which bind tothe chemical moieties, each of which may be induced to emitelectromagnetic radiation of a different wavelength. In one embodimentof the invention, each moiety is attached to a different analyte ofinterest.

EXAMPLES

[0124] This invention is illustrated in the examples which follow. Theexamples are set forth to aid in an understanding of the invention butare not intended to, and should not be construed to, limit in any waythe invention as set forth in the claims which follow thereafter.

Example 1Tris(6,6,7,7,8,8,8-heptofluoro-2,2-dimethyl-3,5-octanedionato)europium

[0125] The sodium salt of2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione was prepared as awhite precipitate by the addition of 0.1M aqueous NaOH (0.7 ml, 0.7 mol)to 2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione (208 mg, 0.7mmol) to form a solution containing the ligand. Absolute ethanol (0.7ml) was then added with stirring and the solution was warmed to 40° C.for 15 minutes to effect complete dissolution of the salt. Eu(NO₃)₃ (100mg, 0.23 mmol) dissolved in a 1:1 water/ethanol mixture (1.0 ml) wasadded to the ligand solution in one portion. A yellow oil formedimmediately. The mixture was stirred at room temperature for one hour.After this time, water (15 ml) was added and the product was extractedwith methylene chloride (20 ml) and dired over anhydrous sodium sulfate.The solvent was then removed under vacuum to afford 220 mg oftris(6,6,7,7,8,8,8-heptofluoro-2,2-dimethyl-3,5-octanedionato)europiumas an oil (92% of theoretical yield). FAB mass spectrometry results wereas follows: m/e, 1077/1075 (M⁺+K), 1061/1059 (M⁺+Na), 1039/1037 (M⁺+1).The product showed identical properties (mass spectrometry, tlc) to thecommercial compound available from Aldrich (Cat. No. 16,039-8, CAS No.17631-68-4).

Example 2 Effect of TPA on Electrochemiluminescence of Eu*

[0126] Cyclic voltammograms were established for 2 mM europium chelatein 0.1 M TBAPF₆ acetonitrile solution and for 2 mM europium chelate with10 mM TPA added in 0.1 M TBAPF₆ acetonitrile solution. Bothvoltammograms were established with a glassy carbon electrode (θ=3 mm)at a scan rate of 0.1 Volts/second. The results are depicted,respectively, in FIGS. 1(a) and 1(b). The results confirm that theelectrochemiluminescence of Eu* is directly caused by the oxidation ofTPA.

Example 3 N-Hydroxysuccinimide Ester Activator

[0127] An electrochemiluminescent rare earth chelate linker containingan N-hydroxysuccinimide (“NHS”) ester can be made by forming europiumbis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate) andseparately synthesizing the key linking ligand,N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate.Reacting these two products together under basic conditions affords thedesired europiumbis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)(N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate).This reaction, which is depicted below, is possible since the rate offormation of the europium complexes is much faster than the rate of NHSester hydrolysis at pH 8-9.

[0128] NHS ester complexes are used to effect linkages to melecules,biomolecules, and biopolymers of intereset that either naturally containprimary or secondary amine functions or that have been modified tocontaine such amines.

Example 4 N-Hydroxysuccinimide Ester Activator

[0129] Alternatively, an electrochemiluminescent rare earth chelatelinker containing an NHS ester can be made by an alternate route,depicted below, in which the NHS ester is formed after the third ligandhas been complexed to the europium.

Example 5 Dicyclohexylcarbodiimide Activator

[0130] An electrochemiluminescent rare earth chelate linker containing adicyclohexylcarbodiimide in place of the NHS ester can be made by boththe procedure of Example 3 and the alternative procedure of Example 4.

[0131] Dicyclohexylcarbodiimide complexes are used to effect linkages tomelecules, biomolecules, and biopolymers of intereset that eithernaturally contain primary or secondary amine functions or that have beenmodified to containe such amines.

Example 6 Amine Activator

[0132] An example of an amine ligand, for linking to electrophilic acylcenters such as NHS esters to form amides or to Michael-type acceptorssuch as maleimides, is depicted below.

[0133] Example 7

Thiol Activator

[0134] An example of a thiol linker, for linking to other thiols,forming thioethers with electrophilic alkyl species such as alkylbromides, or reacting with Michael-type acceptors such as maleimides, isdepicted below.

Example 8 Labelling of Bovine Serum Albumin With Activated EuropiumComplex

[0135] Europiumbis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)(N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate)in dimethylformamide solution is added to a stirred solution of bovineserum albumin (BSA) in aqueous Physiologic Buffered Saline (PBS, 5 ml;25 mg/ml BSA). The mixture is stirred overnight, and precipitate isremoved by centrifugation. The supernatant contains europium-labelledBSA.

Example 9 Labelling of Human Immunoglobulin G With Activated EuropiumComplex

[0136] Europiumbis(1,1,1,2,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate)(N-hydroxysuccinimidyl-9,9,10,10,11,11,11-heptafluoro-6,8-undecanoate)in dimethylformamide solution is added to a stirred solution of humanimmunoglobulin G (IgG) in aqueous buffer. The europium-labelled IgGsolution fluoresces brightly after extensive dialysis, indicating thatthe europium complex is bound to the high molecular weightaffinity-purified human IgG.

[0137] Although the present invention has been described and illustratedwith reference to certain preferred and other specific embodimentsthereof, those skilled in the art will be apprised by the teachingsherein of principles of wide applicability. The invention patented,therefore, is determined only by the scope and spirit of the appendedclaims.

What is claimed is:
 1. A chemical moiety having the formula[MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide element; P is apolydentate ligand of M; L¹ and L² are ligands of M, each of which maybe a substance covalently bound to one or more of P, L¹, or L² throughone or more covalent bond linkages, said linkages designated as (-link-)and being covalent bonds linking B with at least one of P, L¹, or L²,and t is an integer equal to or greater than 1; B is a biologicalsubstance, a synthetic substance which is capable of competing with abiological substance in a competitive binding reaction with acomplementary material, or a non-biological polymer; P, L¹, L², and Bare of such number that the total number of bonds to M provided by theligands of M equals the coordination number of M; and P, L¹, L², and Bare of such composition that the chemical moiety can be induced to emitelectromagnetic radiation.
 2. The chemical moiety of claim 1, wherein Bis a member selected from the group consisting of whole cells,subcellular particles, polypeptides, proteins, nucleic acids,polysaccharides alkaloids, steroids, vitamins, amino acids, plantpathogens, serum-derived antibodies, monoclonal antibodies, and T4thyroid hormones.
 3. The chemical moiety of claim 1, wherein (-link-) isthe linkage formed between one of said ligands and a free amino groupwhich is part of B.
 4. The chemical moiety of claim 1, wherein (-link-)is the linkage formed between a carboxyl group and a free amino groupwhich is part of B.
 5. The chemical moiety of claim 1, wherein M iseuropium or terbium and (-link-) is an amide linkage that covalentlybonds B with a substituted bipyridyl ligand, the amide linkage beingformed by reaction of a carboxyl substituent on said substitutedbipyridyl ligand with a free amino group which is part of B.
 6. Thechemical moiety of claim 1, wherein M is europium and P, L¹, and L² areeach 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato ligands.7. The chemical moiety of claim 1, wherein M is terbium and P, L¹, andL² are each 2,2,6,6-tetramethyl-3,5-heptanedionato ligands.
 8. A methodof determining the presence of a chemical moiety, said chemical moietyhaving the formula [MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; Pis a polydentate ligand of M; L¹ and L² are ligands of M, each of whichmay be a substance covalently bound to one or more of P, L¹, or L²through one or more covalent bond linkages, said linkages designated as(-link-) and being covalent bonds linking B with at least one of P, L¹,or L²; t is an integer equal to or greater than 1; B is a biologicalsubstance, a synthetic substance which is capable of competing with abiological substance in a competitive binding reaction with acomplementary material, or a non-biological polymer; P, L¹, L², and Bare of such number that the total number of bonds to M provided by theligands of M equals the coordination number of M; and P, L¹, L², and Bare of such composition that the chemical moiety can be induced to emitelectromagnetic radiation; which method comprises (a) forming a reagentmixture containing the chemical moiety, or the chemical moiety and anagent which upon exposure of the reagent mixture to electrochemicalenergy forms either a reductant or an oxidant; (b) exposing the reagentmixture to electrochemical energy the potential of which oscillatesbetween a potential sufficiently positive to oxidize the chemical moietyand a potential sufficiently negative to reduce the chemical moiety, orto electrochemical energy such that said chemical moiety is oxidized andthe agent forms a reductant, or such that said chemical moiety isreduced and the agent forms an oxidant, thereby to induce the chemicalmoiety to electrochemiluminescence; and (c) detecting emittedluminescence thereby to determine the presence of the chemical moiety.9. The method of claim 8, wherein M is europium; P, L¹, and L², each isa semi-aromatic oxygen-containing ligand, each of said ligands being thesame or not the same as each other ligand; and (-link-) is one or moreamide linkages, ester or thioester linkages, or ether or thioetherlinkages, each said linkage covalently bonding B with at least one of P,L¹, and L².
 10. A system for determining the presence of a chemicalmoiety, which system comprises (a) a reagent mixture comprising thechemical moiety, or the chemical moiety and an agent which upon exposureof the reagent mixture to electrochemical energy forms either areductant or an oxidant, said chemical moiety having the formula[MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; P is a polydentateligand of M; L¹ and L² are ligands of M, each of which may be asubstance covalently bound to one or more of P, L¹, or L² through one ormore covalent bond linkages, said linkages designated as (-link-) andbeing covalent bonds linking B with at least one of P, L¹, or L²; t isan integer equal to or greater than 1; B is a biological substance or asynthetic substance which is capable of competing with a biologicalsubstance in a competitive binding reaction with a complementarymaterial, or a non-biological polymer; P, L¹, L², and B are of suchnumber that the total number of bonds to M provided by the ligands of Mequals the coordination number of M; and P, L¹, L², and B are of suchcomposition that the chemical moiety can be induced to emitelectromagnetic radiation; (b) means for exposing the reagent mixture toelectrochemical energy the potential of which oscillates between apotential sufficiently positive to oxidize the chemical moiety and apotential sufficiently negative to reduce the chemical moiety, or toelectrochemical energy such that said chemical moiety is oxidized andthe agent forms a reductant, or such that said chemical moiety isreduced and the agent forms an oxidant, thereby to induce the chemicalmoiety to electrochemiluminescence; and (c) means for detecting emittedluminescence thereby to determine the presence of the chemical moiety.11. A method of determining the presence of an analyte of interest whichbinds to a chemical moiety, which comprises (a) forming a reagentmixture comprising the chemical moiety, or comprising the chemicalmoiety and an agent which upon exposure of the reagent mixture toelectrochemical energy forms either a reductant or an oxidant, and theanalyte of interest; such that the chemical moiety and the analytespecifically bind to one another, said chemical moiety having theformula [MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; P is apolydentate ligand of M; L¹ and L² are ligands of M, each of which maybe a substance covalently bound to one or more of P, L¹, or L² throughone or more covalent bond linkages, said linkages designated as (-link-)and being covalent bonds linking B with at least one of P, L¹, or L²; tis an integer equal to or greater than 1; B is a biological substance ora synthetic substance which is capable of competing with a biologicalsubstance in a competitive binding reaction with a complementarymaterial, said biological substance or synthetic substance being capableof specifically binding to the analyte of interest; P, L¹, L², and B areof such number that the total number of bonds to M provided by theligands of M equals the coordination number of M; and P, L¹, L², and Bare of such composition that the chemical moiety can be induced to emitelectromagnetic radiation; (b) exposing the reagent mixture toelectrochemical energy the potential of which oscillates between apotential sufficiently positive to oxidize the chemical moiety and apotential sufficiently negative to reduce the chemical moiety, or toelectrochemical energy such that said chemical moiety is oxidized andthe agent forms a reductant, or such that said chemical moiety isreduced and the agent forms an oxidant, thereby to induce the chemicalmoiety to electrochemiluminescence; and (c) detecting emittedluminescence thereby to determine the presence of the analyte ofinterest.
 12. A composition which comprises (i) a chemical moiety havingthe formula [MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; P is apolydentate ligand of M; L¹ and L² are ligands of M, each of which maybe a substance covalently bound to one or more of P, L¹, or L² throughone or more covalent bond linkages, said linkages designated as (-link-)and being covalent bonds linking B with at least one of P, L¹, or L²,and t is an integer equal to or greater than 1; B is a biologicalsubstance, a synthetic substance which is capable of competing with abiological substance in a competitive binding reaction with acomplementary material, or a non-biological polymer; P, L¹, L², and Bare of such number that the total number of bonds to M provided by theligands of M equals the coordination number of M; and P, L¹, L², and Bare of such composition that the chemical moiety can be induced to emitelectromagnetic radiation; said linkages being covalent bonding linkingB with at least one of P, L¹, or L², and (ii) one or more differentchemical moieties each of which is capable of being induced to luminesceat a wavelength different from that at which any other of the chemicalmoieties luminesces.
 13. A competitive binding method of determining thepresence of an analyte of interest wherein the analyte and a chemicalmoiety bind competitively to a complementary material, which comprises(a) forming a reagent mixture comprising the analyte of interest, thecomplementary material and the chemical moiety, or the chemical moietyand an agent which upon exposure of the reagent mixture toelectrochemical energy forms either a reductant or an oxidant, such thatthe chemical moiety and the analyte of interest bind competitively tothe complementary material; said chemical moiety having the formula[MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; P is a polydentateligand of M; L¹ and L² are ligands of M, each of which may be asubstance covalently bound to one or more of P, L¹, or L² through one ormore covalent bond linkages, said linkages designated as (-link-) andbeing covalent bonds linking B with at least one of P, L¹, or L²; t isan integer equal to or greater than 1; B is a biological substance or asynthetic substance which is capable of competing with a biologicalsubstance in a competitive binding reaction with a complementarymaterial, said biological substance or synthetic substance being capableof binding to the complementary material; P, L¹, L², and B are of suchnumber that the total number of bonds to M provided by the ligands of Mequals the coordination number of M; and P, L¹, L², and B are of suchcomposition that the chemical moiety can be induced to emitelectromagnetic radiation; (b) exposing the reagent mixture toelectrochemical energy the potential of which oscillates between apotential sufficiently positive to oxidize the chemical moiety and apotential sufficiently negative to reduce the chemical moiety, or toelectrochemical energy such that said chemical moiety is oxidized andthe agent forms a reductant, or such that said chemical moiety isreduced and the agent forms an oxidant, thereby to induce the chemicalmoiety to electrochemiluminescence; and (c) detecting emittedluminescence thereby to determine the presence of the analyte ofinterest.
 14. A system for determining the presence of an analyte ofinterest which binds to a chemical moiety, which system comprises (a) areagent mixture comprising the chemical moiety, or comprising thechemical moiety and an agent which upon exposure of the reagent mixtureto electrochemical energy forms either a reductant or an oxidant, andthe analyte of interest; said chemical moiety having the formula[MPL¹L²-(-link-)-]_(t)B wherein M is a lanthanide; P is a polydentateligand of M; L¹ and L² are ligands of M, each of which may be asubstance covalently bound to one or more of P, L¹, or L² through one ormore covalent bond linkages, said linkages designated as (-link-) andbeing covalent bonds linking B with at least one of P, L¹, or L²; t isan integer equal to or greater than 1; B is a biological substance or asynthetic substance which is capable of competing with a biologicalsubstance in a competitive binding reaction with a complementarymaterial, or a synthetic substance being capable of binding to theanalyte of interest; P, L¹, L², and B are of such number that the totalnumber of bonds to M provided by the ligands of M equals thecoordination number of M; and P, L¹, L², and B are of such compositionthat the chemical moiety can be induced to emit electromagneticradiation; (b) means for contacting the chemical moiety with the analyteof interest to form a reagent mixture; (c) means for exposing thereagent mixture to electrochemical energy the potential of whichoscillates between a potential sufficiently positive to oxidize thechemical moiety and a potential sufficiently negative to reduce thechemical moiety, to electrochemical energy such that said chemicalmoiety is oxidized and the agent forms a reductant, or such that saidchemical moiety is reduced and the agent forms an oxidant, thereby toinduce the chemical moiety to electrochemiluminescence; and (d) meansfor detecting emitted luminescence thereby to determine the presence ofthe chemical moiety.
 15. A compound having the formula MPL¹L²-(-link-)wherein M is a lanthanide element; P is a polydentate ligand of M; L¹and L² are ligands of M, each of which may be a substance covalentlybound to one or more of P, L¹, or L² through one or more covalent bondlinkages, said linkages designated as (-link-) and being covalent bonds;P, L¹, and L² are of such number that the total number of bonds to Mprovided by the ligands of M equals the coordination number of M; and P,L¹, and L² are of such composition that the chemical moiety can beinduced to emit electromagnetic radiation.
 16. The chemical moiety ofclaim 15, wherein M is europium and P, L¹, and L² are each6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato ligands. 17.The chemical moiety of claim 15, wherein M is terbium and P, L¹, and L²are each 2,2,6,6-tetramethyl-3,5-heptane-dionato ligands.
 18. Thechemical moiety of claim 15, wherein (-link-) contains anN-hydroxysuccinimidyl moiety
 19. The chemical moiety of claim 15,wherein (-link-) contains an amino moiety
 20. The chemical moiety ofclaim 15, wherein (-link-) contains a thiol moiety